The use of membrane electrophoresis and of electrofiltration as preparative separation techniques has been investigated since the 50 s.
In both methods, separation chambers are separated from one another by semipermeable membranes.
In membrane electrophoresis, migration of dissolved ions and dispersed, charged particles and agglomerates takes place in an electric field, but substantially no liquid flows through the separation membrane. The driving force for the transport of the material is the electric field.
In electrofiltration, a hydrostatic pressure difference is built up between the separation chambers in addition to the electric field, so that a liquid flow through the separation membrane is induced. Driving forces for the transport of material are therefore the electric field as well as the pressure difference between the separation chambers.
The electrofiltration was first mentioned by Bier, 1959, U.S. Pat. No. 3,079,318, as so-called “Forced Flow Electrophoresis”. It was used for reducing the blocking of membranes in the filtration of yeast but also in the depletion of cells and albumin from blood.
U.S. Pat. No. 3,989,613 (Gritzner 1976) describes a membrane electrophoresis method in which the two streams (feed and permeate) flow in parallel. The flow velocity in the separation chambers is very low (<0.01 cm/s). This results in a relatively poor heat discharge and nonturbulent flow, which in turn leads to undesired concentration polarization. Nonselective separation membranes are used.
A further publication on electrofiltration is the dissertation thesis by Tison, 1986, Selective cascade electrofiltration. This is concerned with the separation of IgG from human blood plasma by means of electrofiltration. However, since the IgG-depleted plasma is to be returned to humans, the mode of operation at physiological saline concentration was chosen. The desalination is thus carried out neither before nor during the process, and the mode of operation has constant current and constant pressure. Owing to the high conductivity of the blood plasma, however, only low productivities can be realized.
The method of electrofiltration was further investigated by Perry et al., 1992, U.S. Pat. No. 5,087,338. Perry describes the separation efficiency of electrofiltration by means of protein-containing model solutions with respect to the selectivity. Biomolecules whose isoelectric points have a difference of 0.1 can be separated. In the examples disclosed, the method is operated at constant current. Since the conductivity of the greatly diluted feed solutions is very low and hardly differs from that of the other buffers in the permeate channel and in the electrode compartments, conductivity changes can hardly occur in the feed solution. These are laboratory-scale applications with model substances, which are not relevant in practice. In contrast, changes in the conductivity occur in practice.
In U.S. Pat. No. 5,437,774, of 1993, Laustsen explains the so-called “Electrodialysis of molecules having a high molecular weight”. Starting from the separation technique of dialysis, he describes the possible design of the cell with one or more separation membranes and different chambers for diluate and concentrate. The membranes may be both uncharged membranes and ion exchange membranes. The process can be operated without permeate flow through the membrane (classical membrane electrophoresis) or with a flow forced by pressure, also described below as electrofiltration. This can be operated in the abovementioned design or with a plurality of channels in parallel, with the result that the membrane area and thus the cost-efficiency of the method are increased, as well as by possibly carrying out a plurality of separation steps in succession. In a working example, Laustsen describes the separation of BSA and hemoglobin. The procedure operates at constant voltage and constant pressure (transmembrane pressure of 67 mbar) at a comparatively high voltage (160V) and low transmembrane flow (0.1 to 0.2 m/s). On the industrial scale, this parameter combination would lead to problems with the removal of heat and to concentration polarization of the proteins at the separation membranes.
A patent application of the company Gradipore (1999, WO A 99/62937) entitled “Purification of Antibodies” describes the purification of antibodies, especially monoclonal antibodies, from peritoneal water of mice with the aid of membrane electrophoresis and the so-called Gradiflow technology (AU A 601040). The pH of the solution was adjusted so that the antibody is present in uncharged form and the charged impurities are electrophoretically depleted through the membrane.
In order to carry out the purification of the antibody, i.e. the depletion of the impurities, as completely as possible, the experiment can be interrupted and the concentrate volume exchanged or the experiment can be carried out in two stages, the pH of the solution, the pore size of the membrane and the direction of the electric field being adapted accordingly. The method is carried out at constant voltage of 200 V. On the industrial scale, the resulting high electric field strength would lead to problems with heat removal and to concentration polarization and protein denaturing at the membrane.
A similar method is disclosed in 2004 in DE A 102 534 83. In this application, a laboratory plant which can be scaled up is presented for the first time (receiver volume of 11). By means of a flow velocity of 5.2 cm/s in the separation chambers, sufficiently high heat discharge can be ensured. Membranes form electrically charged double layers at the solid/liquid interface by diffusion of ions out of the membrane into an aqueous medium. The use of porous membranes therefore induces in the electric field an electroosmotic pressure which produces a liquid flow through precisely this membrane. In order to compensate this electroosmotic flow, an exactly tailored electroosmotic pressure is superposed on one of the separation chambers in this method. As a result of the compensation of the electroosmotic flow, the productivity and especially the selectivity of an electrophoretic method can be significantly improved.
Furthermore, DE A 0 40 07 848, “Vorrichtung und Verfahren für die Membranelektrophorese und Elektrofiltration [Apparatus and method for membrane electrophoresis and electrofiltration]”, describes a membrane module with which a protein separation can preferably be carried out by means of membrane electrophoresis or electrofiltration. An advantage here is the closed, tightly joined design, which ensure high transmembrane flow velocities in the separation channels, a minimization of dead spaces and high compactness. The laid-open application also describes the basic use of the modules for electrofiltration. The choice and optimization of the operating parameters, such as, for example, hydrostatic pressure and electric field strength, are not a subject of this laid-open application.
Usually, antibodies are carried out by a certain method explained in more detail in the U.S. Pat. No. 5,429,746, Shadle et al. “Antibody Purification” of 1995.
There are a certain number of purification steps which are used in one or other combination and sequence.
The key step of this method is product recovery and concentration from the fermentation medium. This consists as a rule of concentration and rebuffering by means of ultrafiltration, followed by an affinity chromatography step. The latter is highly selective but has a number of disadvantages on the industrial scale. Examples which will be mentioned are:                the high price of the chromatography medium        the danger of washing out the toxic ligand protein A, which requires an after treatment (as a rule an additional chromatography step)        the low pH in the elution (<3), which entails a further step of rebuffering in order to be able to subject the medium to the following steps of the method.        
Product recovery (capturing) is followed as a rule by at least two further chromatography steps (purification and polishing).
The methods described to date for the purification of antibodies thus have a number of disadvantages:
Classical Methods:
The conventional purification of proteins from a fermentation is effected at least using the above-described number of process steps. These complex processes are associated with high costs, which can be reduced by the novel method according to the invention.
Electrophoretic Methods Generally:
Membrane electrophoresis and electrofiltration have not been carried out to date on the industrial or production scale. In addition, model solutions having a conductivity which is substantially below that of a liquid with physiological saline concentrations, such as, for example, that of a cell culture medium, were chosen in the majority of cases.
Electrofiltration:
Experiments on electrofiltration were operated in the past with either constant current or constant voltage. Since biotechnological media and product solutions in the process have a relatively high conductivity, a reduction thereof during the electrofiltration is required in order to achieve the high mobility in the electric field and hence a high productivity of the process. Thus, in order to be able at all to realize such a process other than on the laboratory scale and other than with a model solution, the following must be noted:
In addition to the productivity, the selectivity is a further target parameter of the electrofiltration process. The selectivity of the separation in turn depends decisively on the pressure-driven transport of material through the separation membrane and on migration velocity of the proteins in the feed channel. Migration of dissolved ions in an electric field can be directed either in the same direction as or in the opposite direction to the pressure-driven transport of material. The target parameters productivity and selectivity can now be optimized for the given process by means of the operating parameters voltage and hydrostatic pressure. The operating parameters are chosen as a function of the physical properties of the dissolved components (for example, isoelectric point, molar mass, effective charge at chosen pH) and of the medium (for example, conductivity). However, if the conductivity of the medium in the feed channel changes, the optimum operating point cannot be maintained with constant operating parameters.
The problem is to be explained for the example of an electrofiltration method in which a monoclonal antibody is to be retained in the feed solution while the secondary components are depleted through the separation membrane. The pH of the solution is chosen so that the monoclonal antibody has a negative charge. Hydrostatic pressure and electric voltage are now chosen so that the migration velocity of the monoclonal antibody in the electric field corresponds at least to the permeate velocity of the medium through the membrane and is in the opposite direction to it. Secondary components which are neutral or have a positive charge are depleted through the separation membrane.
If the solution for the permeate chamber with a physiological conductivity which corresponds to that of the cell culture medium (about 10 mS/cm) is used, the optimum ratio of migration velocity of the antibody and permeation velocity of the medium through the separation medium can be maintained over the entire course of the experiment. The selectivity is therefore constantly optimum during the entire course of the experiment. Owing to the high conductivities and a limited heat discharge, however, only a relatively low productivity can be achieved.
If, however, a solution of low conductivity is used for the permeate chamber in order to desalinate the cell culture medium in the feed channel, the conductivity of the medium in the feed channel in the course of the experiment matches that of the permeate channel. During operation at constant voltage, the migration velocity of the antibody increases owing to the increasing electric field strength in the feed channel. Increasing productivity can, however, hardly be realized at constant hydrostatic pressure. If, on the other hand, the hydrostatic pressure is chosen to be higher from the beginning of the experiment, increased productivity is achieved. However, the selectivity of the method is lower at the beginning of the method and the antibody yield is reduced.
It is therefore the object of the invention to provide a method for purifying charged macromolecules, the operating parameters of which can be adapted so that the target parameters productivity and selectivity are at the chosen optimums over the entire course of the experiment.
The macromolecule-containing, in particular protein-containing, medium, in particular an antibody-containing fermentation medium, is to be concentrated by means of electrofiltration and the secondary components present in the fermentation supernatant are to be separated from the target protein.